This invention relates to an antibody (or fragment or derivative thereof) and preferably, to an antibody (or fragment or derivative thereof) which binds to human lymphocytes. More particularly, this invention relates to preventing and/or inhibiting on-going immune responses in a patient through the administration of such antibody (or fragment or derivative thereof) to a patient. Preferably, this invention relates to preventing or inhibiting T cell activation and proliferation through the administration of such antibody or fragment or derivative thereof to a patient.
The establishment of donor-specific immunological tolerance to primarily vascularized allografts would obviate the use of chronic immunosuppressive drug therapy and the associated morbidity and mortality. The main hurdle in developing tolerance protocols for use in humans is to determine the least toxic but consistently successful immunosuppressive regimens. It is therefore of overwhelming importance to establish experimental models, especially large animal models which will reliably be predictive of use in the clinical setting. Outbred primates seem to be the most appropriate clinical relevant model, and central tolerance induction to primarily vascularized renal allografts has. been previously reported in cynomolgus monkeys. (Kawai, et al., Transplantation, Vol. 59 pg. 256 (1995); Kimikawe, Transplantation, Vol. 64, pg. 709 (1997)). Tolerance induction in this model was based on development of mixed hematopoietic chimerism and was obtained by concomitant donor bone marrow infusion, non-myeloablative total body irradiation (3 Gy) and an immunosuppressive regimen including transient injection of rabbit anti-human thymocyte globulins and cyclosporin. Other models of tolerance induction in primates including post-transplant transient depletion of peripheral T cells with rabbit anti-thymocyte globulins and subsequent infusion of donor bone marrow and total lymphoid irradiation have been reported. (Carver, et al., Transplant Proc., Vol. 23, pg. 480 (1991)). Mixed chimerism seems to be the key for central tolerance induction, but devplopment of microchimerism requires, at least, non lethal myeloablative irradiation combined with T cell-depleting agents, as tolerance seems to be favored by a milieu with minimally reactive T cells. (Contreras, et al., Transplantation, Vol. 65, pg. 1159 (1998)). The use of high doses of total irradiation in the clinical setting is, however, hazardous, especially in children, and efforts must be made in order to establish immunosuppressive protocols using easily monitorable T cell-depleting reagents that result in central tolerance and microchimerism as previously discovered in rodent studies. (Sykes, et al., Nature Medicine, Vol. 3, pg. 783 (1997)).
Recent studies showed that tolerance to primarily vascularized allografts in primates might be induced without irradiation, by using a new anti-CD3 immunotoxin (Contreras, et al. 1998). This very powerful T-cell depleting agent was used in conjunction with donor lymphohematopoietic cell infusion in order to produce a high level of chimerism (Contreras, et al. 1998; Knechtle, et al., Transplantation, Vol. 63, pg. 1 (1997); Thomas, et al., Transplantation, Vol. 64, pg. 124 (1997)). The profound and long-lasting T-cell depletion in both peripheral blood and lymph nodes may, however, limit its clinical applicability. In addition, the pre-existence of an immune agent against the diphtheria toxin moiety of the anti-CD3 immunotoxin could reduce the efficiency of this agent. Nonetheless, this molecule clearly demonstrates that central tolerance induction might be achievable in primate models without total body irradiation.
As of this time, there are no available immunosuppressive agents other than anti-CD3 immunotoxin which might produce such a strong peripheral and lymphoid T cell depletion in humans. Nonetheless, the Campath-1 mAb has been used in human clinical trials for bone marrow transplantation and provided a high incidence of mixed chimerism and encouraging results have been reported in renal allografts using this mAb. (Friend, Ann. Acad. Med. Singapore, Vol. 20, pg. 503 (1991); Nagler, et al., Bone Marrow Transplantation, Vol. 18, pg. 475 (1996); Friend, et al., Transplantation, Vol. 48, pg. 248 (1989); Hamilton, et al., Bone Marrow Transplantation, Vol. 17, pg. 819 (1996)). Recently, another mAb (LO-CD2a/BTI-322) has shown interesting T cell-depleting effects in humans. LO-CD2a/BTI-322, which is an anti-human CD2 mAb has demonstrated great efficiency in preventing acute cellular rejection in renal transplantation (Mourad, et al., Transplant. Proc., Vol. 29, pg. 2353 (1991)), in graft versus host disease and liver transplantation (manuscript in preparation). This mAb, however, does not react with primate T cells, except for human and chimpanzee T cells and therefore cannot be used in experiments involving baboons and cynomolgus monkeys.
In accordance with the present invention, there is provided a monoclonal antibody or fragment thereof that recognizes both baboon and human CD2+ cells. As the CD2 molecule is expressed on all subsets of T cells including CD2 natural killer (NK) cells, LO-CD2b may be used as an immunotherapeutic reagent as well as providing a powerful tool for studies on optimizing conditioning regimens.
More particularly, in accordance with an aspect of the present invention, there is provided a molecule (preferably a monoclonal antibody or fragment thereof) which binds to the same epitope (or a portion thereof) on human lymphocytes as the monoclonal antibody produced by the cell line deposited as ATCC Deposit No. PTA-802. The antibody which is produced by the deposited cell line is hereinafter sometimes referred to as LO-CD2b. The term xe2x80x9cmoleculexe2x80x9d or xe2x80x9cantibody that binds to the same epitope as LO-CD2bxe2x80x9d includes LO-CD2b. The term xe2x80x9cLO-CD2bxe2x80x9d includes the antibody produced by the deposited cell line and those identical thereto which may be produced, for example, by recombinant technology.
The molecules or antibodies of the present invention inhibit human T-cell activation and proliferation and Applicant has found that such inhibition can be effected when adding the molecule or antibody either before or after an agent which stimulates T-cell activation.
The molecules or antibodies of the present invention have the characteristics of binding to an epitope of a CD2 antigen (CD2 positive human T-cells) but it is to be understood, however, that the ability of such molecules or antibodies to inhibit T-cell activation or proliferation may or may not be effected through binding to CD2 positive cells, although Applicant presently believes that the mechanism of action involves binding of the molecule or antibody to CD2 positive cells.
In accordance with another aspect of the present invention there is provided a method of preventing and/or inhibiting on-going immune response in human patients through the administration to the patient of an antibody, hereinafter referred to as LO-CD2b (or fragment or derivative thereof) or any molecule that mimics such antibody or derivative or fragment thereof.
A cell line which produces LO-CD2b, was deposited on Jun. 22, 1999, at the American Type Culture Collection, 10801 University Blvd., Manassas, Va. 20110-2209, and was given the ATCC accession number PTA-802. Such antibody is a rat monoclonal antibody.
Although Applicants do not want to limit the invention to any theoretical reasoning, it is believed that the mechanism which enables the monoclonal antibody of this invention to prevent or reduce the severity of an immune response, and to inhibit the activation and proliferation of T-cells, is the fact that the LO-CD2b antibody either decreases the density of CD2 expressed on T cell surfaces and thus decreases the number of CD2+ T lymphocytes; and/or affects signal transduction. It is believed that these mechanisms of action are responsible for not only the prevention of immune response, but also the reduction in severity of on-going immune responses.
In accordance with an aspect of the present invention there is provided a process for inhibiting initial or further activation and proliferation of T cells in a human patient by administering to the patient an effective amount of a molecule (preferably an antibody) which binds to the same epitope (or any part thereof) on human lymphocytes as the LO-CD2b antibody. The preferred molecule is LO-CD2b or a chimeric and/or humanized form thereof. Such a molecule would, for example, contain the same complementarity determining region (CDR) as the LO-CD2b antibody.
The term xe2x80x9cinhibitxe2x80x9d as used herein throughout this Applicant is intended to mean prevention, or inhibition, or reduction in severity, or induction of tolerance to, or reversal of graft rejection. The term xe2x80x9cgraftxe2x80x9d as used herein for purposes of this application shall mean any and all transplantation, including but not limited to, allograft and xenograft transplantation. Such transplantation may by way of example include, but not be limited to, transplantation of cells, bone marrow, tissue, solid-organ, bone, etc.
The term xe2x80x9cimmune response(s)xe2x80x9d as used herein is intended to mean immune responses dependent upon T cell activation and proliferation which includes both cellular effects and T cell dependent antibodies which may be elicited in response to, by way of example and not limitation: (i) grafts, (ii) graft versus host disease, and (iii) autoantigens resulting in autoimmune diseases, which by way of example include but are not limited to rheumatoid arthritis, systemic lupus, multiple sclerosis, diabetes mellitus, etc.
The molecule employed in the present invention is one which binds to the same epitope (or a part of that epitope) as the LO-CD2b monoclonal antibody. The term xe2x80x9cbinds to the same epitope as LO-CD2b monoclonal antibodyxe2x80x9d is intended to describe not only the LO-CD2b monoclonal antibody but also describes other antibodies, fragments or derivatives thereof or molecules which bind to the same such epitope as the LO-CD2b monoclonal antibody.
Such other antibodies include by way of example and not limitation rat, murine, porcine, bovine, human, chimeric, humanized antibodies, or fragments or derivatives thereof.
The term xe2x80x9cderivativexe2x80x9d as used herein means a chimeric or humanized antibody, single chain antibody, bispecific antibody or other such antibody which binds to the same epitope (or a portion thereof) as recognized by the LO-CD2b monoclonal antibody.
The term xe2x80x9cfragmentxe2x80x9d as used herein means a portion of an antibody, by way of example such portions of antibodies shall include but not be limited to CDR, Fab, or such other portions, which bind to the same epitope or any portion thereof as recognized by LO-CD2b.
The term xe2x80x9cantibodyxe2x80x9d as used herein includes polyclonal, monoclonal antibodies as well as antibody fragments, derivatives as well as antibodies prepared by recombinant techniques, such as chimeric or humanized antibodies, single chain or bispecific antibodies which bind to the same epitope or a portion thereof as recognized by the monoclonal antibody LO-CD2b. The term xe2x80x9cmoleculesxe2x80x9d includes by way of example and not limitation, peptides, oligonucleotides or other such compounds derived from any source which mimic the antibody or binds to the same epitope or a portion thereof as the antibody fragment or derivative thereof.
Another embodiment of the present invention provides for a method of treating a patient who is to receive or has received a graft transplant with an effective amount of at least one member selected from the group consisting of LO-CD2b antibody, or an antibody, or derivative or fragment thereof or molecules which bind to the same epitope (or a portion thereof) as the LO-CD2b antibody. The treatment is preferably effected with the whole or intact LO-CD2b antibody.
A monoclonal antibody of this invention as hereinabove described may be produced by techniques known in the art such as described by Kohler and Milstein (Nature 256, Pg. 495-497, 1975) as well as the techniques disclosed herein. The preparation of a monoclonal LO-CD2b antibody is described in more detail in Example 1 of this Application. As hereinabove indicated LO-CD2b antibodies may also be produced by recombinant techniques using procedures known in the art. The recombinant antibody may also be in the form of a chimeric antibody wherein the variable regions of a LO-CD2b rat antibody are combined with the constant region of an antibody of another species. Thus, for example, the monoclonal antibody may be humanized by combining the CDR regions of a rat LO-CD2b monoclonal antibody with the V region frameworks and constant regions of a human antibody to provide a chimeric human-rat monoclonal antibody.
In one embodiment, the antibody is a humanized form of LO-CD2b antibody constructed from the constant regions of a human antibody, and the framework and CDR regions of the light and heavy chain variable regions, in which the framework regions of the light and heavy chain variable regions are derived from the framework regions of the light and heavy chain variable region of a human antibody, and the CDR""s are the rat LO-CD2b CDR""s. In one embodiment, one or more amino acid residues of the framework regions of the light and heavy chain variable regions may be amino acid residues from the rat LO-CD2b framework regions. Such residues from the rat framework regions are retained in the humanized antibody because such residues may maintain the binding specificity of LO-CD2b. Thus, in producing a humanized antibody, in accordance with a preferred aspect of the invention the CDR""s of a human antibody are replaced with the CDR""s of LO-CD2b with the added factor that certain amino acids of the light chain variable portion of LO-CD2b in particular from FR1, FR2 and FR3 and certain amino acids of the heavy chain variable portion of LO-CD2b in particular from FR-2 and FR-3 are retained in constructing the humanized antibody; i.e., the corresponding. amino acids of the human framework are replaced with the noted amino acids from the rat LO-CD2b framework.
In another embodiment, the present invention is related to a chimeric antibody comprised of a human constant region and the variable regions from rat LO-CD2b and to the use thereof.
The preparation of LO-CD2b monoclonal antibody suitable for the purposes of the present invention should be apparent to those skilled in the art from the teachings herein.
An antibody or fragment or derivative thereof or molecule of the type hereinabove described may be administered in vivo in accordance with the present invention to inhibit the activation and proliferation of T-cells, and decrease the density of CD2 expression on the cell surface and thereby reduce the number of CD2+ T lymphocytes.
Thus, for example, in an in vivo procedure, such LO-CD2b antibodies are administered to prevent and/or inhibit immune response and thereby inhibit T cell activation and proliferation.
An antibody or fragment or derivative thereof or molecule of the type herein above described may be administered ex vivo in accordance with the present invention to decrease the density of CD2+ expression on the cell surface and thus reduce the number of CD2+ cells of the donor cells. By way of example and not limitation, in an ex vivo procedure, such antibodies or fragments or derivatives thereof or molecules would be infused into donor bone marrow prior to transplantation to prevent the onset of graft versus host disease upon transplantation.
In such an in vivo or ex vivo technique, the antibody or fragment or derivative thereof or molecule will be administered in a pharmaceutically acceptable carrier. As representative examples of such carriers, there may be mentioned normal saline solution, buffers, etc. Such pharmaceutical carriers are well known in the art and the selection of a suitable carrier is deemed to be within the scope of those skilled in the art from the teachings contained herein.
The LO-CD2b antibody or other molecule of the present invention may be administered in vivo intravenously or by intramuscular administration, etc.
As herein above indicated, LO-CD2b antibody or other molecule of the present invention is administered in vivo in an amount effective to inhibit graft rejection. The term xe2x80x9can effective amountxe2x80x9d for purposes of this Application shall mean that amount of monoclonal antibody capable of producing the desired effect, i.e., the inhibition of graft rejection or inhibition of the activation of T-cells. In general, such antibody is administered in an amount of at least 1 mg. It is to be understood that lower amounts could be used. In addition after the initial treatment, the herein above described amounts may be reduced for subsequent treatments, if any. Thus the scope of the invention is not limited by such amounts.
In accordance with the present embodiment, such antibodies are administered in order to maintain the inhibition of T-cell activation and graft rejection. Thus, by way of example and not limitation, the antibody may be administered by intravenous infusion over a one to two hour period in amount of from about 1 mg/dose to about 50 mg/dose in a physiologically acceptable carrier suspension once or twice a day for a period of from about eight. days or more, as needed. Such treatment for graft rejection is preferably started at, or immediately prior to, or shortly after transplantation or when graft rejection occurs. The treatment could be given once or twice a day for as little as one or two days when. started at the time of transplantation to induce a selective hyporesponsive state to the transplant. Such treatment for autoimmune diseases with respect to the administration of the antibody or fragment or derivative thereof or molecule in accordance with the present invention is begun when the attending physician has determined it is desirable to inhibit a pathological immune response.
Thus, in accordance with an aspect of the present invention, by administering an antibody in accordance with the invention at the time of transplantation and in most cases for a short period thereafter there can be induced a hyporesponsiveness to the transplanted tissue or organ, thereby to prevent or inhibit further episodes of rejection.
The techniques of the present invention for inhibiting the activation of T-cells may be employed alone or in combination with other techniques, drugs or compounds for inhibiting the activation of T-cell or inhibiting graft rejection or graft versus host disease.
The invention will be further described with respect to the following examples, which are illustrative and which are not intended to limit the scope of the invention.
The cells, cultures, mAbs and mitogens used in the examples may be prepared and used by processes and procedures known and practiced in by those of ordinary skill in the art. The following are examples of processes or procedures that may be used for the preparation and use of the cells, cultures, mAbs and mitogens used in the examples which follow.
The purpose of the studies shown in Examples 1 through 6 was to determine whether LO-CD2b could inhibit human and nonhuman primate immune response in vitro and nonhuman immune response in vivo. The in vivo T cell depletion was therefore assessed in both peripheral blood and lymph nodes. In addition to being a strong immunosuppressive agent in vitro, LO-CD2b demonstrated-the ability to prolong a renal allograft survival.